Integrin from human epithelial cells

ABSTRACT

Novel hybridoma cell lines producing monoclonal antibodies which react specifically with human pancreatic cancer cells are described. Methods for producing antigenic preparations to generate the hybridoma cell lines and for selecting, purifying and characterizing the monoclonal antibodies reactive with human cells, including pancreatic cancer cells, are disclosed. The antigens to which the antibodies of the invention are specific are characterized.

This is a continuation of copending application Ser. No. 07/293,384filed on Jan. 4, 1989, now abandoned, which is a continuation-in-part ofU.S. Ser. No. 016,552, filed Feb. 19, 1987, by Kajiji and Quafanta,which is now abandoned and incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to a novel antigen and to novelhybridoma cell lines, and more specifically to monoclonal cell linesproducing monoclonal antibodies reactive with the novel antigen, whichantigen can be found on human pancreatic cancer cells.

Cancer currently constitutes the second most common cause of death inthe United States. Carcinomas of the pancreas are the eighth mostprevalent form of cancer and fourth among the most common causes ofcancer deaths in this country. The incidence of pancreatic cancer hasbeen increasing steadily in the past twenty years in most industrializedcountries, exhibiting the characteristics of a growing epidemiologicalproblem.

The prognosis for pancreatic carcinoma is, at present, very poor, itdisplays the lowest five-year survival rate among all cancers. Suchprognosis results primarily from delayed diagnosis, due in part to thefact that the early symptoms are shared with other more common abdominalailments. The diagnosis of pancreatic cancer is often dependent onexploratory surgery, inevitably performed after the disease has advancedconsiderably.

Substantial efforts have been directed to developing tools useful forearly diagnosis of pancreatic and other carcinomas. Nonetheless, adefinitive diagnosis is often dependent on exploratory surgery which isinevitably performed after the disease has advanced past the point whenearly treatment may be effected. One promising method for earlydiagnosis of various forms of cancer is the identification of specificbiochemical moleties, termed antigens present on the surface ofcancerous cells. Antibodies which will specifically recognize and bindto the antigens present on the surfaces of cancer cells potentiallyprovide powerful tools for the diagnosis and treatment of the particularmalignancy. Tumor specific cell surface antigens have previously beenidentified for certain melanomas, lymphomas malignancies of the colonand reproductive tract.

There thus exists a great and long-felt need for a cell surface markerwhich is present on the surface of neoplastic cells, including those ofthe pancreas, and for antibodies which specifically recognize such acell surface marker. Such markers and corresponding antibodies would beuseful not only in the early detection of pancreatic and other cancers,but in their treatment as well. The present invention satisfies theseneeds and provides related advantages as well.

Cell adhesion is critical to many biological processes, includingembryonal development, tissue repair, immune response, and malignanttransformation. (Ekblom, P., et al. (1986) Ann. Rev. Cell. Biol.2:27-47; Yamada, K. M. (1983) Ann. Rev. Biochem. 52:761-799; Edelman, G.M. (1983) Science 219:450-457.) Several laboratories have recently donebiochemical characterization of adhesion receptors for extracellularmatrix and plasma proteins such as fibronectin and vitronectin as wellas leukocyte adhesion receptors. (Tamkun, J. W., et al. (1986) Cell46:271-282; Damsky, C. H., et al. (1981) J. Cell. Biol. 89:173-184;Pytela, R., et al. (1985) Cell 40:191-198; Fitzgerald, L. A., et al.(1987) J. Biol. Chem. 262:3936-3939; Giancotti, F. G. (1985) Ext. CellRes. 156:182-190; Springer, T. A. (1985) Nature 314:540-542.) Theseadhesion receptor proteins have been shown to be structurally homologousto each other. (Charo, I .F. (1986) Proc. Natl. Acad. Sci. USA83:8351-8355; Suzuki, S. (1986) Proc. Natl. Acad. Sci. USA 83:8416-8418;Kishimoto, T. K. (1987) Cell 48:681-690; Takada, Y. (1987) Nature326:607-609.) These related molecules have now been organized into aprotein superfamily, designated "integrins", after the chickenfibronectin/laminin receptor. (Hynes, R. O. (1987) Cell 48:549-554.)

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies characterized inthat the antibodies react specifically to human pancreatic carcinoma(HPC) cells. These monoclonal antibodies, which recognize and bind tocell surface markers in HPC cells, may be advantageously used fordiagnosis and treatment of HPC.

In accordance with the present invention there are provided monoclonalantibodies which react specifically with antigenic markers on thesurface of HPC cells. In accordance with a further aspect of theinvention, there are provided hybridoma cell lines which producemonoclonal antibodies specifically reactive with HPC cell surfacemarkers.

Preferred hybridoma cell lines are those termed S3-41 and S3-53identified by ATCC accession numbers HB 9318 and HB 9319, respectively.The monoclonal antibodies produced and the antigens recognized by thesecell lines are also a part of the present invention.

It will be appreciated from the foregoing that the present inventionprovides novel markers for antibodies against HPC tumor cells. In oneaspect of the invention, the monoclonal antibodies are used for in vitroimmunoassays to detect HPC. In another aspect of the invention, themonoclonal antibodies, conjugated with certain detectable labels, areuseful as in vivo imaging agents for detecting HPC. Moreover, whenconjugated with certain toxins, such monoclonal antibodies are usefulfor therapeutic treatment of HPC.

Another aspect of the invention concerns the cell surface markersreactive with the antibodies of the invention. These markers are usefulin characterization of the cells bearing them and in design of agonistsand antagonists of their functions. The antigens reactive with HB 9318antibodies are members of the integrin super family.

The antigen reactive with HB 9318 is a new member of the integrinsuperfamily. As with other integrins, this molecule is a heterodimercomprised of structurally unrelated subunits, both of which areglycosylated. That this integrin, isolated from human epithelial cells,is novel was shown by amino acid sequence homologies. No obviousserologic cross-reactivities were detected with other integrins. Theβ-chain of the epithelial integrin has a molecular weight which issignificantly higher than other integrin β-chains. It is postulated thatthis is due to a large sialic acid content.

Because integrin heterodimers are grouped into three families, basedupon which of the three β-chains they contain, it is proposed that theβ-chain of the present invention be designated β4 in recognition of itsdefining a fourth integrin β-chain family.

Other features and advantages of the present invention will becomeapparent from the following detailed description which illustrates, byway of example, the principles of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows two-dimensional gel analysis of immunoprecipitates obtainedby reacting HB 9318 with radiolabeled extracts of FG cells.

FIG. 2 shows two-dimension gel analysis of immunoprecipitates obtainedby reacting HB 9319 with radiolabeled extracts of FG cells.

FIGS. 3A and 3B show amino acid residue sequences of α-chain, gp125 andβ-chain, gp150 purified using HB 9318.

FIG. 4 shows a comparison of N-terminal sequences of various integrin α-and β-chains.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. DEFINITIONS

"Monoclonal antibodies (Mabs) reactive with HPC" refers to homogenouspopulations of immunoglobulins which are capable of immunoreaction withantigens expressed on human pancreatic cancer (HPC) cells. It isunderstood that there may be a number of antigens present on the surfaceof any cell and, alternatively, that certain receptors present on HPCcells may also occur on other malignant or normal cell types. Moreover,such antigens may, in fact, have a number of antigenic determinants. Theantibodies of the invention may be directed against one or more of thesedeterminants. Any characteristic antigen associated with HPC may providethe requisite antigenic determinant.

Immunoglobulins, like all proteins, may exist in acidic, basic orneutral form depending on their amino acid composition and environment,and may be found in association with other molecules such as saccharidesor lipids. The immunoglobulins of the present invention fall within thedefinition regardless of status in this regard as long as they remaincapable of selectively reacting with HPC associated antigens.

"Cells" or "cell line" refers to the cells apparently denoted as well asthe progeny thereof. It is known that during cell multiplication andgrowth cells or cell lines may not remain precisely constant in theirgenetic makeup and the progeny may, indeed, be distinguishable in someway from the parent cells. So long as the cells referred to hereinretain the characteristic of secretion capability for Mabs reactive withHPC, as defined above, they are to be considered included in thedefinition.

"Immortalizing cell line" refers to a cell line which can be maintainedperpetually, for practical purposes, in cell culture, i.e., for anindefinite number of transfers. It must also, when fused to an ordinarynon-transformed cell line, which would normally not survive for morethan a few days or weeks as a unicellular culture, be able to confer onthe fusion product its own immortal properties.

II. HYBRIDOMA PREPARATION A. GENERAL DESCRIPTION OF HYBRIDOMAPREPARATION

The examples below describe the preparation of specific hybridoma celllines producing monoclonal antibodies reactive with HPC cell antigens.It will be appreciated, however, that alternative methods may beemployed to obtain alternative embodiments of the specific Mabs reactivewith HPC cell antigens.

Techniques for preparing hybridomas are generally well-known in the art.Generally speaking, such hybridoma cell lines are prepared by a processinvolving the fusion under appropriate conditions of an immortalizingcell line and a B lymphocyte cell line appropriately immunized toproduce the desired antibody. While the immortalizing cell lines so usedare often of murine origin, those of any other mammalian species may beemployed alternatively including those of rat, bovine, canine, humanorigin and the like. The immortalizing cell lines are most often oftumor origin, particularly myeloma cells, but may also include normalcells transformed with, for example, Epstein Bart Virus. Anyimmortalizing cell here may be used to prepare the hybridomas of thepresent invention.

Cells capable of secreting antibodies were employed as fusion partners,such as spleen cells or peripheral blood lymphocytes. The animal fromwhich the cells were to be derived was immunized at intervals with wholecell suspensions of human pancreatic cancer cells. Alternatively, cellextracts or purified antigen may be used for immunization.

The immortalizing cells and lymphold cells were fused to form hybridomasaccording to standard and well-known techniques employing polyethyleneglycol as a fusing agent. Alternatively fusion may be accomplished byelectrofusion. Hybridomas are screened for appropriate monoclonalantibody secretion by assaying the supernatant or protein purified fromthe ascites for reactivity with the desired cell or antigen. Such assaytechniques include, among others, ELISA, RIA Western Blotting, orimmunoprecipitation.

In the present invention, hybridomas were initially screened forproduction of antibodies reactive with HPC cells. Alternatively, HPCcell extracts or purified antigens could be used for screening. In orderto further characterize the monoclonal antibodies, their reactivity withvarious HPC cell lines, other tumor cell lines and a variety of othernormal, malignant and non-malignant pathological human tissues wasdetermined using standard assay techniques such as ELISA, RIA,immunoprecipitation, histochemical staining procedures includingindirect immunoperoxidase or indirect immunofluorescence staining. Thehybridomas of the present invention were found to produce monoclonalantibodies generally highly reactive with all human pancreatic celllines. They also displayed high reactivity with cells derived from othertumors, noticeably those of gastrointestinal and genitourinary tractorigin. Moreover, although they displayed some reactivity with certainnormal tissues, the Mab displayed negligible reactivity with majororgans such as liver and kidney. Apparently, the antigens against whichthe antibodies are directed are highly expressed on HPC cells but onlymoderately or less on other cell types.

Because of their selective reactivity with HPC cell derived antigens themonoclonal antibodies are useful for both diagnosis and therapy of HPCand other carcinomas. Moreover, their non-reactivity with liver andkidney cells in particular permits them to be used therapeutically withrelatively little risk of targeting these critical organs.

The antibodies of the invention are also useful in the preparation ofcell surface markers reactive with them using immunoprecipitation and/oraffinity chromatography of cell membrane preparations. The markerreactive with HB 9318 antibodies described below is a member of theintegrin super family, and is functional in mediating binding to theextracellular matrix. Accordingly, this marker is involved in metastasisand colonization of malignant cells.

B. PARTICULAR HYBRIDOMA EMBODIMENTS

The following examples illustrate a method for preparing hybridomaswhich can serve as sources for the desired monoclonal antibodies, andthe antibodies thus produced. While the methods described are typical ofthose which might be advantageously used, other alternative proceduresknown to those skilled in the art may be alternatively employed. Theexamples are thus intended to illustrate, but not to limit theinvention.

EXAMPLE 1 PREPARATION OF HYBRIDOMA CELL LINE

Murine Mabs reactive with HPC cell lines were produced essentiallyaccording to the standard techniques of Kohler and Milstein, Nature256:495 (1975). Briefly, standard HPC cell lines such as COLO 357 andits subclones were used to obtain the antigenic preparation. Preferablycells of the cell line termed FG were employed. (Kajiji, S. M.,Intraneoplastic Diversity in Human Pancreatic Cancer, Ph.D. Thesis,Brown University (1984). Alternatively other pancreatic cell linesexpressing the antigens may be used, such as BxPC-3 (ATCC No. CRL1687).The cells were grown in a monolayer culture and harvested by EDTAtreatment. Briefly, confluent monolayers were incubated for 20 minutesat 37° C. with PBS containing 10 mM EDTA and 0.02% KCl. The detachedcells were collected, centrifuged at 1000 ×g for 10 minutes and washedtwice with cold PBS. Alternatively, cell suspensions were derived fromFG xenografts grown in Balb/c athymic nude mice.

Two to four month old normal Balb/c mice were immunized with whole cellsuspensions at weekly intervals with six intraperitoneal 0.5 mlinjections, containing approximately 5×10⁶ to 5×10⁸cells/injection/mouse. Three days after the final injection, the micewere sacrificed and the spleens removed. The spleens were placed inserum free Dulbecco's Minimal Essential Medium (DMEM) in separate Petridishes and washed. The splenocytes were gently teased out of the fibroussplenic capsule using a rubber policeman. The cell suspension was thenplaced in a 15 ml tube and centrifuged at 1000 ×g for 10 minutes. Thepellet was then washed twice with serum-free DMEM.

The washed spleen cells and the P3X63Ag8 myeloma cells were fusedaccording to the method of Kohler and Milsrein, supra. The immortalizedcell line fusion partners used were the murine myeloma cell lineP3X63Ag8 (ATCC Accession No. T1B9) These myeloma cells were grown at adensity of 5×10⁵ cells/ml and harvested by centrifugation at 1000 ×g for10 minutes. The cell pellet was washed twice with serum free DMEM.Finally, the spleen cells and the P3X63Ag8 myeloma cells were combinedat a ratio of 7:1 in a 50 ml tube and pelleted by centrifugation (1000 gfor 10 minutes). The pellet was gently loosened and 1 ml of a 35%polyethylene glycol (PEG) solution was gently bubbled over the cells.After 1 minute, 1 ml of DMEM, containing 10% fetal calf serum(FCS)(Gibco, Grand Island, N.Y.) was added to the cell suspension andgently mixed.

The PEG was subsequently diluted by the addition of 10 ml DMEMcontaining 10% FCS and the cells were repelleted. The cell pelletcontaining hybridoma fusion products was resuspended in 30 mlhypoxanthine-aminopterin-thymidine (HAT) medium (aminopterin from SigmaChemical Co., St. Louis, Mo.; hypoxanthine and thymidine fromCalbiochem, La Jolla, Calif.).

This cell suspension was then combined with 400 ml of HAT mediumcontaining 2×10⁶ thymocytes per ml (feeder cells). The contents weredistributed into sterile 96 well plates (Costar, Cambridge, Mass.) andplaced immediately in an incubator at 37° C. The spent media wasreplaced with fresh thymocyte containing HAT media after one week. Usingthis type of protocol successful hybridoma cultures were obtained whichcould be maintained with periodic addition of fresh DMEM containing 10%FCS.

Hybridomas producing monoclonal antibodies reactive with HPC cells wereselected. After the cultures reached a cell density that covered 75-100%of the microtiter well surface, media from the hybridomas were screenedfor the presence of anti-HPC antibody, using a standard ELISA protocol.(Schultz, Cancer Res. 44:5914(1984)). Briefly, tumor cells dried ontothe bottom of 96-well miniplates (Dynatech Microtiter Plates, AmericanScientific Products, McGaw Park, Ill.) were used as targets. The wellsof antigen-coated 96 well plates to be used were rinsed with buffer A pH8.0 (20 mM Tris, containing 150 mM NaCl, 0.2% Tween 20 and 0.01%Thimerosal). The hybridoma supernatant diluted 1:2 in buffer B (buffer Acontaining 0.1% bovine serum albumin) was added to the wells andincubated for 1 hour at room temperature to permit binding of specificantibodies. Specifically bound antibodies were detected by addinghorseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin(BioRad, Richmond, Calif.) to wells that were rinsed free of the excesshybridoma supernatant by washing with buffer A. After incubation for 1hour at room temperature the secondary antibody was decanted, the wellswashed with buffer A, and 50 μl/well of substrate solution (tenmilliliters of 80 mM citrate phosphate buffer, pH 5.0 containing 4 mgO-phenylenediamine (Sigma Chem. Co., St. Louis, Mo.) and 4 μl 30%hydrogen peroxide) was added. The plates were incubated in the dark for30 min at RT and the color reaction was stopped by adding 25 μl of 4Msulfuric acid to each well. Specifically-bound antibodies were detectedby measuring the absorbance at OD 490 on an ELISA scanner C model EL310,Biotek Instruments Winooski, Vt.) within 30 min. Reactivity was gradedas follows: A₄₉₀ ≦0.15, -; A₄₉₀ =0.15 to 0.3, 1+; A₄₉₀ =0.3 to 0.6, 2+;A₄₉₀ =0.6 to 1.2, 3+; A₄₉₀ ≧1.2, 4+. Hybridomas that were reactive withthe immunizing FG cells but not with the lymphoblastoid 721-P cells werefurther screened for reactivity with frozen sections of HPC according tothe procedure of Example II below. Only those that were reactive withfrozen sections of HPC but not reactive with frozen sections of normalhuman liver, kidney and lung were selected. The two hybridoma cellslines selected for further study were designated HB 9318 and HB 9319,respectively.

III. CHARACTERIZATON OF MONOCLONAL REACTIVITY EXAMPLE 2 A. REACTIVITYWITH HUMAN TUMOR TISSUES.

The reactivity of the monoclonal antibodies was determined by indirectimmunoperoxidase staining as follows. Two- to 4-μm sections of frozentissue blocks were cut on a cryotome, mounted on gelatin-coated glassslides, air-dried, and tested immediately in an indirectimmunoperoxidase assay using the method of Taylor, Arch. Pathol. Lab.Med. 102:113 (1970). Briefly, after washing once in Hanks' balanced saltsolution (Gibco, Grand Island, N.Y.) and phosphate buffered saline (PBS10 mM sodium phosphate, 0.15M Nacl, pH 7.0), sections were incubated atroom temperature sequentially with: diluting buffer (PBS containing 5%normal goat serum and 1% bovine serum albumin) for 15 min; a 1:2dilution of hybridoma supernatants or appropriate isotype-matchedcontrols for one hour; horseradish peroxidase-conjugated goat anti-mouseIg antiserum (Bio-Rad, Richmond, Calif.) diluted 1:50 and containing 5%normal human serum for one hour; and finally substrate buffer (10 mMTris, pH 7.4, 0.6 mg/ml 3,3'-diaminobenzidine, 0.015% H₂ O₂) for 15 min.Washes with HBSS and PBS were performed between incubations. Sectionswere counterstained in 1% methylene blue, dehydrated through gradedethanol, washed in Histo-Clear (National Diagnostics, Somerville, N.J.),mounted in Pro-Texx (Lerner Laboratories, New Haven, Conn.), andexamined by light microscopy.

Table 1 summarizes the reactivity of monoclonal antibodies produced byhybridoma cell lines HB 9318 and HB 9319 with 65 different tumors. HB9318 was generally reactive only with carcinomas of the pancreasgastrointestinal tract, genitourinary tract, and head and neck tumors.Moreover, in virtually all instances, staining by the HB 9318 Mabs wasdistinctly associated with the basement membranes surrounding tumorfoci, producing a characteristic one-sided basal surface staining ofcells at the epithelial stromal interface. In the few cases of lungcarcinomas, melanoma and breast cancer tissues that were stained,reactivity was also confined to the basement membranes.

Mab HB 9319 reacted with each of the seven pancreatic adenocarcinomastested, including pancreatic carcinoma of the acinar cell type. Mab HB9319 displayed a wide range of reactivity among tumor tissues examined.Moreover, reactivity of HB 9319 was generally intense with the majorityof tumor cells within a tissue. Tumor cell basement membranes were alsostained in some cases.

                  TABLE 1                                                         ______________________________________                                        REACTIVITY.sup.a OF MONOCLONAL                                                ANTIBODIES WITH FRESH FROZEN                                                  HUMAN TUMOR TISSUE SECTIONS BY                                                IMMUNOPEROXIDASE STAINING                                                                       HB 9318  HB 9319                                            ______________________________________                                        Pancreatic Cancer                                                             ductal adenocarcinoma                                                                             -.sup.b    2+                                                                 2+.sup.b   1+                                                                 2+.sup.b   4+                                                                 -          1                                                                  4+.sup.b   1+                                                                 3+.sup.b   3+                                                                 4+         4+                                             islet cell Cancer/insulinoma                                                                      -          -                                                                  -          1+                                             acinar cell Cancer  -          3+                                             Oral Squamous Cancer                                                                              1.sup.b    3+                                                                 1.sup.b    4+                                                                 -          2+                                                                 -.sup.b    4+                                             Adenoid Cystic Cancer                                                                             3+         2+                                             Salivary Gland Cancer                                                                             4+.sup.b   3+                                             Esophageal Cancer   3+.sup.b   3+                                             Gastric Cancer      3+.sup.b   3+                                                                 2+         4+                                                                 3+         3+                                                                 1+.sup.b   3+                                             Colon Cancer        3+.sup.b   3+                                                                 -.sup.b    1+                                                                 3+         3+                                                                 3+.sup.b   4+                                             Hepatoma            -          2+                                                                 -          2+                                             Laryngeal Cancer    2+.sup.b   4+                                                                 2+.sup.b   3+.sup.b                                       Melanoma            1+.sup.b   4+                                                                 -          4+                                                                 -          4+                                             Sarcoma             -          4+                                                                 -          4+                                             Lung Cancer                                                                   adenocarcinoma      1+         1+                                                                 -          3+                                                                 -          1+                                             squamous Cancer     1+.sup.b   3+                                                                 -          2+                                                                 -          3+                                                                 -          3+                                                                 -          3+                                             adenosquamous       1+.sup.b   3+                                             oat cell Cancer     -          -                                                                  -          -                                              large cell Cancer   4+         4+                                             mesothelioma        -          1+                                             Breast Cancer       -          3+                                                                 -          4+                                                                 -          3+                                                                 -          2+                                                                 1+         2+                                             Cervical Cancer     2+.sup.b   1+                                                                 1+         4+                                             Endometrial Cancer  -          3+                                                                 -          4+                                                                 3+.sup.b   2+                                             Ovarian Cancer      -          4+                                                                 -          3+                                                                 3+.sup.b   2+                                             Prostatic Cancer    1+.sup.b   4+                                             Bladder Cancer      1+.sup.b   2+                                                                 3+.sup.b   2+.sup.b                                       Kidney Cancer       -          4+                                                                 2+         -                                              ______________________________________                                         .sup.a Intensity of staining was scored from 1+ to 4+ with 4+ indicating      greatest intensity and with "-" indicating lack of staining.                  .sup.b Basement membrane staining.                                       

B. REACTIVITY WITH HUMAN CELL LINES

The reactivity of Mabs against a panel of cell lines in culture wasdetermined by ELISA reactivity, according to the Method of Schultz(1984), Cancer Res. 44:5914, as detailed in Example 1

Cells dried onto the bottom of 96-well miniplates were used as targetsfor ELISA. Horseradish peroxidase-conjugated goat anti-mouse Igantiserum (BioRad, Richmond, Calif.) was used as the secondary antibody.

The reactivity of Mabs HB 9318 and HB 9319 is shown in Table 2. BothMabs were reactive with the majority of the ten HPC cell lines tested.Moreover, both displayed particularly strong reactivity with cell linesderived from lung cancer, skin cancer and gastrointestinal andgenitourinary tract tumors. HB 9319 displayed moderate to strongpositivity with tumor cell lines of neuroectodermal origin includingmelanoma, glioblastoma and neuroblastoma lines. Both antibodies weregenerally non-reactive with human red blood cells of blood types AB+,A+, B+, O+, and O-, normal diploid fibroblasts and leukemic or lympholdcell lines.

                  TABLE 2                                                         ______________________________________                                        ELISA REACTIVITY OF MONOCLONAL ANTIBODIES                                     WITH CULTURED HUMAN CELLS                                                     Cell lines (ATCC NO.)                                                                             HB 9318    HB 9319                                        ______________________________________                                        Pancreatic Cancer                                                             Colo 357.sup.a      3+         2+                                             FG.sup.a            3+         3+                                             SG.sup.a            3+         3+                                             FG-Met-2.sup.a      4+         4+                                             RWP-1.sup.a         4+         2+                                             RWP-2.sup.a         3+         1+                                             PANC-1 (CRL 1469)   3+         3+                                             ASPC-1 (CRL 1682)   3+         1+                                             Hs 766T (HTB 134)   4+         1+                                             BxPC-3 (CRL 1687)   4+         4+                                             Lung Cancer                                                                   adenocarcinoma                                                                UCLA-P3.sup.b       -          2+                                             A549 (CCL 185)      2+         1+                                             CALU 6 (HTB 56)     -          4+                                             squamous cancer                                                               T-222.sup.b         2+         4+                                             SK-MES-1 (HTB 58)   3+         3+                                             CALU-1 (HTB 54)     2+         3+                                             USCLS-1.sup.b       3+         3+                                             oat cell Cancer                                                               T-293.sup.c         -          1+                                             NCI-H69 (HTB 119)   -          -                                              Breast Cancer                                                                 734B.sup.d          -          3+                                             BT-20 (HTB 19)      3+         4+                                             MDA-MB-435S (HTB 129)                                                                             -          3+                                             Bladder Cancer                                                                T24 (HTB 4)         -          2+                                             J82 (HTB 1)         -          2+                                             5637 (HTB 9)        2+         3+                                             Cervical Cancer                                                               ME-180 (HTB 33)     3+         4+                                             Prostatic Cancer                                                              DU-145 (HTB 43)     2+         3+                                             Pharyngeal Cancer                                                             FaDu (HTB 43)       3+         4+                                             Skin cancer                                                                   A-431 (CRL 1555)    3+         3+                                             Colon Cancer                                                                  COLO 396.sup.d      4+         4+                                             Hepatoma                                                                      SK-HEP-1 (HTB 52)   3+         2+                                             Mesodermal Tumor                                                              SK-UT 1 (HTB 114)   -          2+                                             Melanoma                                                                      ML-873-1.sup.c      -          2+                                             WM239A.sup.c        -          3+                                             WM2664 (CRL 1676)   -          3+                                             A375P.sup.c         -          4+                                             A-375M.sup.c        -          3+                                             M14.sup.c           -          3+                                             M21.sup.c           -          4+                                             MS-1.sup.c          -          3+                                             FOSS.sup.c          -          3+                                             Melur.sup.c         -          3+                                             Glioblastoma                                                                  U38MG (HTB 16)      -          1+                                             U87MG (HTB 14)      -          3+                                             U-373MG (HTB 17)    -          3+                                             Neuroblastoma                                                                 SK-N-SH (HTB 11)    -          2+                                             SK-N-MC (HTB 10)    -          -                                              LAN-1.sup.c         2+         1+                                             B-Lymphoblastoid                                                              L14.sup.b           -          1+                                             LG-2.sup.b          -          1+                                             721-P.sup.e         -          -                                              GM3107.sup.b        -          2+                                             T-Lymphoblastoid                                                              MOLT-4 (CRL 1582)   -          -                                              HPB-ALL.sup.b       -          2+                                             HSB-2.sup.d         -          -                                              Promyelocytic Leukemia                                                        HL-60 (CCL 240)     -          -                                              Erythroleukemia                                                               K562 (CCL 243)      -          -                                              Diploid Fibroblast                                                            W1-38 (CCL 75)      -          -                                              Human RBC           -          -                                              ______________________________________                                         Cell lines were obtained as follows:                                          .sup.a P. Meitner, Department of Medicine, Brown University                   .sup.b L. Walker, Department of Immunology, Scripps Clinic and Research       Foundation                                                                    .sup.c R. Reisfeld, Department of Immunology, Scripps Clinic and Research     Foundation                                                                    .sup.d T. Edginton, Department of Immunology, Scripps Clinic and Research     Foundation                                                                    .sup.e F. Bach, University of Minnesota.                                 

C. REACTIVITY WITH NON-MALIGNANT PATHOLOGIC HUMAN TISSUES

The reactivity of the Mabs with a panel of inflammatory pancreases,benign tumor and hyperplastic epithelia was determined by indirectimmunoperoxidase staining of frozen tissue sections, according to themethod of section A, above. Both Mab HB 9318 and Mab HB 9319 showed somereactivity with the duct cells of chronic pancreatitis tissues. Mab HB9319 was widely reactive in that it stained every non-malignantpathologic tissue examined, although always in discrete areas. Table 3shows the results of testing with this panel of tissues.

                  TABLE 3                                                         ______________________________________                                        REACTIVITY OF MONOCLONAL ANTIBODIES WITH                                      FRESH FROZEN NON-MALIGNANT                                                    PATHOLOGIC HUMAN TISSUE                                                       SECTIONS BY IMMUNOPEROXIDASE STAINING                                                        HB 9318   HB 9319                                              ______________________________________                                        Pancreas (chronic pancreatitis)                                               acini            -       -       -     -                                      ducts            2+      -.sup.b 2+    1+                                     islets of Langerhans                                                                           -       -       -     -                                      Pancreas (SLE).sup.a                                                          acini              -           4+                                             ducts              -.sup.b     -                                              islets of Langerhans                                                                             -           -                                              Uterus (leiomyoma)                                                                             - -     - -     3+ 4+ 4+ 4+                                  Ovary (fibroadenoma)                                                                             -           2+                                             Endometrium (hyperplastic)                                                                       3+          4+                                             Prostate (hyperplastic)                                                       upper layers of epithelium                                                                       -           3+                                             basal layers of epithelium                                                                       4+          3+                                             basement membrane  4+          -                                              ______________________________________                                    

D. REACTIVITY WITH NORMAL ADULT AND FETAL TISSUES.

The reactivity of the Mabs with fresh frozen normal adult and fetaltissues was determined by indirect immunoperoxidase staining accordingto the method of section A, above. The antibodies were unreactive withthe vast majority of normal tissues examined.

Mab HB 9318 displayed some reactivity with the basal epithelial layersor basement membranes of the esophagus, cervix, and large intestine,plantar skin, breast tissue and ileal epithelium. The restrictedexpression of the HB 9318 antigen by the proliferating cell layers ofnormal stratified epithelia and its localization at the epithelialstromal interface suggests that this molecule may be an earlydifferentiation antigen (possibly involved in cell adhesion) ofepithelial cells that is re-expressed following malignanttransformation. Further, the HB 9318 antigen may be useful for diagnosisand therapeutic intervention of other skin-related disorders such aspsoriasis and basal cell carcinomas and may prove to be a valuable cellsurface marker for investigating epidermal cell biology.

Mab HB 9319 reacted with the acinar cells of adult and fetal pancreases,fetal pancreatic ducts, and the parenchyma and bile ducts of 1/3 liversthat were tested. It was moderately reactive with the esophagus, stomachand small intestine, cervix, uterus, breast, fetal and adult lungparenchyma, fetal kidney, cerebral cortex, and with the molecular layersand Purkinje cells within the adult cerebellum. All layers of plantarskin including basement membrane were also intensely stained.

Table 4 summarizes the results of this panel of tests.

                  TABLE 4                                                         ______________________________________                                        REACTIVITY OF MONOCLONAL ANTIBODIES                                           WITH FRESH FROZEN NORMAL HUMAN TISSUE                                         SECTIONS BY IMMUNOPEROXIDASE STAINING                                                        HB 9318  HB 9319                                               ______________________________________                                        Esophagus                                                                     stratified squamous epithelium                                                upper layers     -          3+                                                basal layers     4+         4+                                                basement membrane                                                                              4+         -                                                 Stomach                                                                       gastric pits     -          3+                                                gastric glands                                                                parietal cells   -          2+                                                chief cells      -          2+                                                lamina propria   -          1+                                                Small Intestine                                                               jejunal epithelium                                                                             -          2+                                                ileal epithelium 3+         3+                                                basement membrane                                                                              4+         -                                                 Large Intestine                                                               colonic epithelium                                                                             -          1+                                                crypts of Lieberkuhn                                                                           -          1+                                                basement membrane                                                                              4+         -                                                 lamina propria   -          1+                                                Liver                                                                         parenchyma       - - -      - 3+ -                                            bile ducts       - 1+ -     - 3+ -                                            Pancreas (adult)                                                              acini            - - - -    4+ 4+ 4+ 4+                                       ducts            - - -.sup.a -                                                                            - - - -                                           islets of Langerhans                                                                           - - - -    - - - -                                           Pancreas (fetal)                                                              acini            -          4+                                                ducts            -.sup.a    4+                                                islets of Langerhans                                                                           -          -                                                 Thymus                                                                        cortex           -          -                                                 medulla          -          -                                                 Lymph node                                                                    nodules          -          -                                                 germinal centers -          1+                                                Spleen                                                                        white pulp       -          -                                                 red pulp         -          1+ 1+                                             Kidney (adult)                                                                glomeruli        - - -      - - -                                             proximal tubules - - -      - - -                                             distal tubules   - - -      - - -                                             Kidney (fetal)                                                                glomeruli        -          3+                                                proximal tubules 1+         3+                                                distal tubules   -          3+                                                Cervix                                                                        columnar epithelium                                                                            - 1+       4+ 3+                                             basement membrane                                                                              4+ 4+      - -                                               squamous epithelium                                                           upper layers     - -        3+ 3+                                             basal layers     4+ 4+      4+ 4+                                             basement membrane                                                                              4+ 4+      - -                                               Uterus                                                                        endometrium      1+ -       3+ 2+                                             myometrium       - -        4+ 2+                                             Ovary                                                                         cortex           -          -                                                 medulla          -          -                                                 Breast                                                                        lobule           4+ 2+      4+ 4+                                             duct             4+ 3+      4+ 4+                                             basement membrane                                                                              4+ 4+      4+ 4+                                             Lung (adult)                                                                  parenchyma       - - -      2+ 2+ -                                           Lung (fetal)                                                                  parenchyma       -          3+                                                Thyroid                                                                       epithelial cells -          1+                                                colloid          -          -                                                 Cerebrum                                                                      cortex           -          3+                                                Cerebellum                                                                    granular layer   -          -                                                 molecular layer  -          2+                                                Purkinje cells   -          2+                                                Plantar skin                                                                  stratum corneum  -          3+                                                stratum granulosum                                                                             -          3+                                                stratum spinosum 1+         3+                                                stratum germinativum                                                                           3+         4+                                                basement membrane                                                                              4+         4+                                                ______________________________________                                         .sup.a Basement membrane staining                                        

E. REACTIVITY WITH CELL SURFACES

To determine whether the antigens recognized by the Mabs were expressedon surface of cells of reactive tissues, viable HPC cells were testedwith the Mabs in indirect immunofluorescence assays as follows:

INDIRECT IMMUNOFLUORESCENCE STAINING

Cells grown to confluence on glass cover slips were washed once withcold HBSS, overlaid with 0.1 ml of 1:2 hybridoma supernatant for onehour at 4° C., washed in cold HBSS, and overlaid with 0.1 ml of 1:50fluorescein isothiocyanate-conjugated goat anti-mouse Ig antiserum(Tago, Burlingame, Calif.) for one hour at 4° C. After washing andfixing in 3% paraformaldehyde, cells were mounted in 80% glycerol, 1mg/ml p-phenylenediamine, 200 mM Tris, pH 8.5, examined and photographedwith a Zeiss fluorescence microscope.

Both Mab HB 9318 and HB 9319 showed distinct staining of the plasmamembrane, indicating recognition of cell surface structures. Bothstained the entire cell population, displaying a contiguous, linearmembrane pattern.

IV. ANTIGEN CHARACTERIZATION EXAMPLE 3 IMMUNOCHEMICAL CHARACTERIZATIONOF ANTIGENS

In order to assess the chemical nature of the antigens recognized byMabs, HPC cells were radiolabeled by incubation with either L-[³ H]leucine or [³ H] glucosamine, detergent solubilized and then subjectedto immunoprecipitation with Mab immunosorbents, as follows:

Ten μl of a 10% suspension of protein-A-Sepharose (Pharmacia, Uppsala,Sweden) were incubated at 4° C. for 1 hour with 5 μl of rabbitanti-mouse Ig antibodies (Accurate Chemicals, Westbury, N.Y.) in 0.3 mlof PORT buffer (10 mM Tris, pH 8.5, 0.15M NaCl, 0.5% Tween 20, 0.1%Renex 30, 2.5 mM sodium azide, 0.1% ovalbumin). After washing twice withPORT buffer, incubating 1 hour at 4° C. with 1 ml hybridoma supernatantsand washing twice with PORT buffer, the beads were incubated overnightat 4° C. with radiolabeled cell extract ((1-2×10⁷ cpm). Theimmunosorbents were washed 8 times with PORT buffer (10 mM Tris, pH 8.5,0.15M NaCl, 0.5% Tween 20, 0.1% Renex 30, 2.5 mM sodium azide) and boundantigens were eluted in Laemmli buffer (Nature 227:680(1970)). Thesamples were analyzed by SDS-PAGE on slab gels and visualized byfluorography.

Initial results of the SDS-PAGE analysis indicated that Mab HB 9318recognized a doublet protein antigen, of 205 kd and 135 kd,respectively. Both bands were glycosylated as they incorporated [³H]-glucosamine. In some cases two additional bands of 150 kd and 185 kdwere also seen. A band of 116 kd co-precipitated with HB 9318 bands, butwas non-specific since it could be removed by preabsorption with controlimmunosorbants. Mab HB 9319 recognized a highly glycosylated 140 kdprotein.

Immunoprecipitation of a metabolically labeled HPC indicated that theantigenic determinants recognized by each of the monoclonals are carriedby protein molecules. These proteins are also glycosylated, so that itremains to be determined whether the recognized epitopes are expressedby the protein or the glycan part of these molecules.

EXAMPLE 4 CHARACTERISTICS OF HB 9318 ANTIGEN

Characterization of the antigen which immunoprecipitates with HB 9318demonstrates that it is a two-chain heterodimer which is a member of theintegrin class of cell adhesion receptors.

Integrins are heterodimers comprised of noncovalently associatedtransmembrane glycoproteins. (reviews of the characteristics ofintegrins have been published by Hynes, R. O., Cell (1987) 48:549-554;Ruoslahti, E., et al, Science (1987) 238:491-497; and Buck, C. A. &Horwitz, A. F. (1987) Ann. Rev. Cell. Biol. 3:179-205. The α-chains havehigh degrees of homology and the differences in the β-chain serve toplace the various integrins into subfamilies. Integrin heterodimers aregrouped into three families, based on which of the three β-chains (β₁,β₂ or β₃) they contain. The integrin of the present invention isbelieved to represent a fourth member of the integrin family because ofits structurally distinct β-chain.

The integrin class of cell surface adhesion receptors is distinct fromanother type designated CAMs which are monomers which usepolysialylation as a control element. The integrin to which HB 9318binds is distinguished from other known integrins by itspolysialylation. The designation of α_(E) β₄ has been proposed as adesignation for this novel integrin. Alternatively, the name intepsinhas been proposed for this antigen.

The HB 9318 antigen contains two non-covalently bound glycopeptides,designated herein gp205 and gp125. The gp125 represents an analog to theα-chains of other integrins, the gp205 peptide is analogous to otherintegrin β-chains, and has been shown to be polysialylated. Thistwo-chain antigen has been shown by immunoperoxidase staining to beexpressed only on the basolateral surfaces of the germinative layer ofepithelial cells while cells from the prickle-cell layer outwards wereprogressively devoid of reactivity. The integrin is evidently involvedin cell adhesion.

When HB 9318 monoclonal antibody is used to immunoprecipitate pancreaticcancer cell lysates gp125 and gp205 are precipitated. When HB 9318monoclonal antibody is used to immunoprecipitate placental cell lysates,gp125 is again precipitated, however a second gp150 β subunit isprecipitated. This gp150 appears to be identical to the gp205 β subunitpresent in pancreatic cancer, with the exception that it is not asglycosylated.

A. DISTRIBUTION OF THE ANTIGEN AND α- AND β-CHAINS

Immunohistology of human epidermal sections with HB 9318 shows thatstaining is concentrated near the basement membrane and basolateral cellsurfaces of the germinative layer. Upper cell layers are progressivelydevoid of reactivity. Thus its expression is restricted to thisparticular portion of the cell surface.

FG-met2 pancreatic carcinoma cells were used as test substrates; lungadenocarcinoma lines and short term normal human keratinocyte culturesgave identical results.

Cultures of FG-met2 pancreatic carcinoma cells were surfaceradioiodinated using ¹²⁵ -I sodium iodide in standard protocols.Detergent lysates of these labeled cells were immunoprecipitated with HB9318, and the immunoprecipitate applied to polyacrylamide gels underreducing and non-reducing conditions. Under reducing conditions, a 205kd band was detected; under non-reducing conditions the single bandappeared at 190 kd. This represents the β-chain of intepsin, designatedherein gp205.

The α-chain, which migrates as a 150 kd band under non-reducingconditions, and a 125 kd band under reducing conditions was detectableonly when the cells were metabolically labeled either with [³⁵S]-methionine or with tritiated glucosamine. It is believed that theabsence of the 125 kd/150 kd bands from the surface-labeled cells is dueto an artifact of the iodination procedure.

That gp205 and gp125 components are non-covalently associated with eachother at the cell surface was verified by treating FG-met2 cells (ahuman pancreatic carcinoma line) with a membrane impermeablecross-linker, DTSSP, and lysing the cells with detergent. This resultedin a 400 kd band upon SDS-PAGE. The 400 kd band disappeared and wasreplaced by 205 kd and 125 kd by subjecting the preparation toreduction, as DTSSP results in reversible cross-linking. Thenon-covalent complexing of gp205 and gp125 was further confirmed byimmunoprecipitating protein from FG-met2 which had been labeled with [³⁵S]-methionine. The immunoprecipitate showed an approximate MW of 500 kdas analyzed by gel filtration. (The discrepancy in apparent MW is anartifact of the procedures.)

B. PURIFICATION OF qp205 AND qp125

The surface proteins were isolated both from the lung adenocarcinomacell line UCLA-P3 and from FG-met2.

The cells were grown in sufficient quantity to obtain 50 g wet weightand 20 g wet weight respectively. After washing, the cells were lysed inan equal volume of TBS containing 2% Renex 30, centrifuged at 10,000 ×gfor 30 min at 4° C. and stored at -70° C.

The lysates were passed sequentially through a Sepharose column andthrough one or two sequential HB 9318 immunosorbent columns at 5-10ml/hr. After washing with TBS containing 0.1% Renex 30, pH 8.5 to removeunadsorbed material, the HB 9318 column was inverted, washed with 3column volumes of TBS, pH 8.5 containing 1.0% n-octylβ-D-glucopyranoside, and the bound material eluted with 50 mMdiethylamine, pH 11.5 containing 150 mMNaCl, 1.0% n-octylβ-D-glucopyranoside. Eluted material was collected in 1.5 ml Eppendorftubes containing 0.1M Tris HCl pH 6.8, 150 mM NaCl, and 1.0% n-octylβ-D-glucopyranoside, to lower the pH rapidly to approximately 8.5.Elution was detected by SDS-PAGE stained with silver stain or Coomassieblue, and eluate from peaks containing protein was pooled andconcentrated.

C. AMINO ACID SEQUENCING OF PURIFIED ANTIGEN

Antigen corresponding to gp205 and to gp125, purified as above wassubjected to amino acid sequencing from the N-termini using standardtechniques. The gp125 fragment had the N-terminal sequence:

    F-N-L-D-T-R-E-D-N-V-I-R-K-Y-G-D-P-G-S-L-F

which shows extensive homology with α N-terminal sequences of α-chainsin other integrins, as illustrated in FIG. 4.

The gp150 fragment had the N-terminal sequence:

    N-R-C-K-K-A-P-V-K-S-C-T-E-C-V-Y-V-D-P

which shows extensive homology with β N-terminal P47 sequences ofβ-chains in other integrins, as illustrated in FIG. 4.

In addition, FIGS. 3A and 3B show the amino acid residue sequences ofthe gp125 α-chain and the gp150 as precipitated by the HB 9318 antibody.

The gp205 β-chain is apparently blocked at the N-terminus.

Automated microsequencing of gp125, purified on an HB 9318immunoaffinity column from either carcinoma cells or placental tissuesyielded information up to residue 21. The sequences of gp125 from thesetwo sources were identical, with the exception of position 6 which couldnot be assigned for carcinoma gp125 in three separate sequencing runs.The gp125 sequence showed several similarities with integrin α chainN-termini, as depicted in FIG. 4. The first five residues, FNLDT, areidentical in four α chains, and occur with one replacement in four otherα chains and with two replacements in the remaining three α chains.Another segment of significant homology among α chains occurs betweenresidues 15 and 21. In this region, gp125 shares one residue with atleast one other α chain at five positions, and at three positions hasunique residues. Overall, the homology of gp125 to the integrin α chainsappears to be equivalent to the homology of the α chains to each other.The highest level of similarity was observed with the VLA-2 α chain,where eleven of thirteen residues available for comparison are shared.

Placenta gp150 was sequenced up to residue 19 (FIG. 3B). When thissequence was compared to the amino-termini of the three human integrin βchains, several similarities were found (FIG. 4). Of particularimportance is the exact correspondence of the three cysteines, theserine in position 10 and the proline in position 19, as these residuesare invariant among integrin β chains. Overall, gp150 showed elevenidentities with β₁, eight with β₂ and seven with β₃. However, the gp150sequence was distinct from those of the other β chains since itsN-terminus was offset with respect to β₁ (as predicted from cDNAs) andβ₃ sequences, and since it contained six unique residues (FIG. 4).

To further check the relationship between gp150 and gp180, placentalgp180 was also sequenced up to residue 13. This sequence was identicalto gp150 (not shown), except for four residues of uncertain assignment,thus providing further evidence for the structural similarity of gp150and gp180. Since the neuraminidase digestions suggested that the M_(r)differences between gp205, gp180 and gp150 are due to variations insialic acid content, gp205 was expected to display an amino-terminalsequence identical to that of gp150 and gp180. However, gp205 isundetectable in placental lysates, and two attempts to sequencecarcinoma-derived gp205 failed, presumably due to insufficientquantities and/or N-blocking during purification.

D. METABOLIC LABELING WITH [³⁵ S]-METHIONINE IN THE PRESENCE OFTUNICAMYCIN

Indirect immunoprecipitation with Mab HB 9318 of detergent lysates ofcells intrinsically labeled with [³⁵ S]-methionine in the presence oftunicamycin (an inhibitor of N-linked glycosylation) and subsequentanalysis by SDS-PAGE under reducing conditions revealed the presence oftwo major bands of 190 kd and 100 kd, respectively.

Semiconfluent cultures of FG cells were incubated for 10-12 hours at 37°C. with 1 μg/ml tunicamycin. The cells were then pulsed for anadditional 12 hours with 1 mCi [³⁵ S]-methionine in methionine-free RPMImedium containing 3% FCS and 1 μg/ml tunicamycin. Control flasks,similarly labeled with [³⁵ S]-methionine in the absence of tunicaymcin,were also prepared. The metabolically-labeled cells were then harvestedas previously described and lysed using RIPA lysis buffer. Cell lysateswere clarified by centrifugation at 100,000 ×g for 45 min at 4° C. andsubsequent storage was at -70° C.

E. TWO-DIMENSIONAL GEL ANALYSES

Immunoprecipitations were carried out by overnight incubation of celllysates with immunoabsorbents prepared by activated-CNBr conjugation ofMab to Sepharose 4B-CL beads (Pharmacia, Uppsala, Sweden). After elutionin 8M urea at room temperature, samples were analyzed by two-dimensionalelectrophoresis, consisting of nonequilibrium pH electrophoresis on tubegels in the first dimension, followed by SDS-PAGE on 7.5 % acrylamideslab gels. Gels were impregnated with 2, 5,-diphenyloxazole, dried andexposed for the indicated times to Kodak XAR-5 X-ray film at -70° C. Theresulting pattern of migration is shown in FIG. 1.

F. CHARACTERIZATION OF THE qp205 AS A POLYSIALYLATE INTEGRIN β-CHAIN

Both gp205 and gp125, alone or together were treated with variousenzymes which cleave carbohydrate chains.

EndoF and EndoH cleave N-linked complex oligosaccharides, and theresults were consistent with one or two such oligosaccharides on gp205and three or four on gp125: ##STR1##

Neuraminidase (NA) cleaves complex N-linked carbohydrates and polysialylchains; endo-N-acetylneuraminidase (Endo-N) cleaves polysialylates onlyas α-2,8-linked linear sialic acid homopolymers. The results wereconsistent with gp205 as a polysialylated integrin β-chain: ##STR2##

These results are consistent with a heavily polysialylated β integrinchain wherein the sialylation is through β-linkage. (Most integrinβ-chains have MW of 95-125 kd).

G. BINDING TO LECTINS

When radiolabeled lysates were preabsorbed with lentil lectin-agarosebeads (Vector Labs, Burlingame, Calif.), the antigens reactive with HB9318 were removed. Removal of antigen was shown by immunoprecipitationof bead supernatant, followed by SDS-PAGE. Similar treatment ofradiolabeled lysates with wheat germ agglutinin-agarose beads (E.Y.Labs., San Mateo, Calif.) did not remove the HB 9318 antigen. Therefore,the antigen reactive with HB 9318 was characteristically bound to lentillectin, but not to wheat germ agglutinin.

H. GEL FILTRATON OF ANTIGEN

Radiolabeled detergent cell lysates were absorbed on wheat germagglutinin-Sepharose columns (E.Y. Labs, San Meteo, Calif.), Thebreakthrough was absorbed onto lentil lectin-Sepharose columns. Afterwashing, the absorbed material was eluted by the addition of 2%alpha-methyl-mannoside (Sigma Chemical Co., St. Louis). The elutedmaterial was subjected to gel filtration, in the presence of 10 mM Tris,pH 8.0 containing 0.15M NaCl, 1 mM CaCl₂, mM MgCl₂.6H₂ O, 0.02% sodiumazide and 0.1% Renex 30 by using an FPLC instrument (Pharmacia, Uppsala,Sweden), equipped with a Sepharose 6 column. Molecular weight standardswere run in parallel. One ml fractions were collected (approximately 40fractions), and subjected to immunoprecipitation with HB 9318 antibody.SDS-PAGE analysis of immunoprecipitates revealed that the followingmolecular species were reactive with HB 9318 in the indicated fractions:

    ______________________________________                                        SDS-PAGE m.w.                                                                             Fraction # FPLC MW                                                ______________________________________                                        135; 205    25         669 kd                                                 135; 205    26         669 kd                                                 135; 150; 205                                                                             27         approximately 669 kd                                   135; 150; 205                                                                             28         approximately 669 kd                                   135; 150; 205                                                                             29         between 669 kd and 440 kd                              135; 150    30         between 669 kd and 440 kd                              135; only   31-36      between 440 kd and 232 kd                              ______________________________________                                    

The estimated molecular weights corresponding to the FPLC fractions areindicated in the right column. Since the estimated MW exceeds the MWdetermined by SDS-PAGE, the HB 9318 antigens must exist as multimericcomplexes, probably heterodimers formed by the association of one 205 kdcomponent with one 125 kd component. The presence of excess free 125 kdcomponent was also suggested by the material immunoprecipitated fromfractions 31-36.

I. PULSE CHASE BIOSYNTHETIC STUDIES:

Single cell suspensions of exponentially growing FG cells werepropagated for one hour at 37° C. in methionine-free medium (IrvineScientific, Santa Ana, Calif.) and then pulse labeled for 10 min with[³⁵ S]methionine (1295 Ci/mM NEN Research Products, Boston, Mass.) at aconcentration of 1.0-1.5 mCi/3×10⁷ cells/mi. After the removal of analiquot of 5×10⁶ cells that constituted the zero-time point, theremaining cells were washed three times with cold Tris-buffer, pH 7.5containing 10 mM unlabeled L-methionine (Sigma Chemical Co., St. Louis,Mo.). The labeled cells were resuspended in complete medium containing10 mM unlabeled methionine and incubated on a shaker at 37° C. Aliquotswere removed at the different time points indicated and the cells werecentrifuged and extracted in RIPA lysis buffer as previously described.

After a 10 minute pulse with [³⁵ S]-methionine, a faint band of 150 kdwas detected at the zero time point of chase. This 150 kd component wasclearly visible after 15 minutes of chase. Within the next 45 minutes ofchase it appeared to be processed and the appearance of the 135 kdcomponent was seen. Both the 205 kd and 135 kd molecules were detectableafter 4 hours of chase until up to 20 hours of chase. It is presentlynot clear whether a precursor/product relationship exists between thetwo forms of the HB 9318 antigen. While not wishing to be bound by theexplanation, it appears that post-translational processing of the 150 kdmolecule gives rise to the 135 kd subunit. Moreover, the 205 kdcomponent could either arise by further processing of 135 kd componentor by altered processing of the 150 kd precursor. Alternatively, itcould have its own precursor molecule that is not recognized by Mab HB9318, thereby suggesting that the HB 9318 antigen is a heterodimercomprised of two distinct non-covalently linked subunits.

Western blots of material immunopurified with HB 9318 from carcinomacells and placental tissue in which 5710 antiserum reacted predominantlywith gp205, gp180, and gp150 and displayed little, if any, reactivitywith gp125. This antigenic similarity of gp205, gp180, and gp150 wasauthentic since, in a further refinement, antibodies that were adsorbedand eluted from gp150 reacted with gp180 (in addition to gp150 itself).Antibodies adsorbed and eluted from gp180 reacted with gp150 (inaddition to gp180 itself). Furthermore, ^(S) 35-methionine labeledgp205, gp180, gp150 and gp125 purified by immunoaffinity andelectroelution were subject to "hot" blotting with gp150 purifiedantibodies. These antibodies only reacted with gp205, gp180, and gp150,but not with gp125. Subsequent autoradiography of the blot confirmedthat the four proteins were present in approximately equal amounts.These results provided further evidence for the structural relatednessfor gp205, gp180, and gp150, and for the dissimilarity of gp125.Therefore, co-precipitation of gp125 by HB 9318 and 5710 antibodies isprobably due to noncovalent associations with gp205, gp180 and gp150.

J. LIMITED PEPTIDE MAPPING

Limited peptide mapping was used to determine the structuralrelationships between gp205 and gp125. Cells labeled with [³⁵S]-methionine were immunoprecipitated with HB 9318 and subjected toSDS-PAGE. The bands to be analyzed were located on the dried gel byautoradiography. These bands were then excised from the gel, rehydratedand inserted in slots on a 15% acrylamide SDS-PAGE gel containing a 0.5microgram/ml solution of Staphylococcus aureus V8 protease (Cleveland,et al. (1977) J. Biol. Chem. 252: 1102-1106.) After the bands werestacked, electrophoresis was suspended for 30 min. to allow enzymaticdigestion. The gels were thereafter fluorographed.

SDS-PAGE resolved eight peptides for gp205 and ten peptides for gp125.Each generated distinct profiles, suggesting that gp205 and gp125 arestructurally unrelated. HB 9318 precipitates two minor bands, gp180 andgp150. Limited peptide mapping of gp180 resolved seven fragments; gp150resolved six fragments. The fragments of both gp180 and gp150co-migrated with gp205 fragments, except for one gp150 peptide whichco-migrated with the major fragment of gp125. (Due to its significantlygreater intensity, this fragment may be derived from minor contaminationof gp150 with the closely-spaced gp125.) These data suggest that gp205,gp180, and gp150 are structurally related, but distinct from gp125.

This conclusion was further supported by investigations with apolyclonal antiserum (5710) raised against antigen purified fromcarcinoma cells by immunoaffinity chromatography on the monoclonalantibody HB 9318. Antiserum 5710 was prepared by bleeding of an NZWrabbit, subcuraneously injected with approximately 2 micrograms ofimmunopurified carcinoma HB 9318 antigen at day 0 in Freund's completeadjuvant, and at days 30 and 45 with the same amount of antigen inincomplete adjuvant.

From carcinoma lysates, the 5710 antiserum precipitated a set ofproteins identical to those reactive with HB 9318. If, however, the celllysates were previously treated at 100° C. in the presence of SDS todisrupt noncovalent associations, then antiserum 5710 precipitated onlygp205, gp180 and gp150, but not gp125. The isolated gp205, gp180 andgp150 displayed their characteristic M_(r) downshift under nonreducingconditions. These results suggest that at least some of the epitopespresent on gp205, gp180 and gp150 are not found on gp125 and that all5710 epitopes on gp125, if any, are sensitive to denaturation. Thisconclusion was verified by Western and "hot" blots.

K. Western and "hot" Blots

The general procedure of Towbin, H. et al. (1979) Proc. Nat'l. Acad.Sci. USA 76:4350-4354, was followed. Proteins separated by SDS-PAGE on a5% gel were transferred overnight at 4° C. to Immobiolon (MilliporeCorp., Bedford Mass.) using a 25 mM Tris, 192 mM glycine buffer. Thefilter was saturated with 3% (w/v) nonfat dry milk in TBS, pH 8.0 with0.05% Tween 20 and 0.02% azide, incubated for 3 h with primary antibody,washed, overlaid for 1 hour with alkaline phosphatase-conjugated goatanti-rabbit (Promega Biotec, Madison, Wis.) or anti-mouse (BoehringerMannheim, Indianapolis, Ind.) IgG, washed again and then developed with0.33 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCL pH9.5, 100 mM NaCl, 5 mM MgCl₂, followed by 5 mM EDTA in 20 mM Tris-HCl pH8.0. For affinity purification (Weinberger, C., et al. (1985) Science228:740-742) of 5710 antibodies, vertical strips cut from the edges ofgp205, gp125 whole-gel blots previously incubated with 5710 weredeveloped to locate reactive bands. Horizontal strips corresponding tothese were excised from the undeveloped mid-part of the filter. Boundantibodies were eluted by three one-minute washes with 5 mM glycine-HClpH 2.3 containing 150 mM NaCl, 0.05% Tween 20, 100 micrograms/ml bovineserum albumin, 2.5 mM sodium azide, and quickly neutralized by additionof Tris-HCl pH 8.0.

For "hot" blots, 1×10⁷ cells were metabolically labeled with [³⁵S]-methionine, immunoprecipitated with HB 9318 and electrophoresed on a5% SDS-PAGE gel. The wet gel was autoradiographed overnight to locateradioactive bands, which were then excised. Proteins were electroelutedin an ISCO apparatus as described (Hunkapiller, M .W., et al (1983)Methods Enzymol. 91:399-413), run on a 5% SDS-PAGE and transferred toImmobilon filters. The filters were first immunostained and thenautoradiographed.

EXAMPLE 5 ADDITIONAL BIOCHEMICAL CHARACTERISTICS OF HB 9319 ANTIGEN A.MOLECULAR PROFILE UNDER NON-REDUCING CONDITIONS

FG-met2 cells labeled with [³⁵ S]-methionine were immunoprecipitatedusing HB 9319 and analyzed by SDS-PAGE under non-reducing conditions. HB9319 antigen migrates as a single band of 125 kd under these conditions.

B. EXTRINSIC RADIOLABELING WITH ¹²⁵ I

Cultures of FG-met2 cells were surface iodinated using ¹²⁵ -I sodiumiodide, and detergent lysates were immunoprecipitated with HB 9319. Theprecipitated antigen migrates on SDS-PAGE under reducing conditions as asingle species of 140 kd.

Similar results were obtained when the antigen was labeled metabolicallywith labeled inorganic sulfate or phosphate.

C. METABOLIC LABELING WITH [³⁵ S]-METHIONINE IN THE PRESENCE OFTUNICAMYCIN

The method follows that of Example 4(D) using HB 9319 in place of HB9318. HB 9319 immunoprecipitated a single band in SDS-PAGE of 100 kdunder reducing conditions.

D. TWO-DIMENSIONAL GEL ANALYSIS

The method follows that of Example 4(E) using HB 9319 in place of HB9318. The resulting pattern of migration is shown in FIG. 2.

E. TREATMENT WITH GLYCOLYTIC ENZYMES

The antigen recognized by HB 9319 is isolated by immunoprecipitation ofradiolabeled cell lysates. The immunoprecipitates are treated with avariety of exoglycolytic and endoglycolytic enzymes. After treatment theapparent mobility of the precipitated antigens is determined in aSDS-PAGE system. The glycolytic enzymes characteristically modify theapparent molecular weight of the HB 9319 antigens, by removing discreteportions of O-linked or N-linked glycans. The results obtained can beschematically summarized as follows:

    ______________________________________                                        Enzyme     Apparent MW After Treatment                                        ______________________________________                                        Endo H     140 kd                                                             NA         135 kd                                                             Endo N     140 kd                                                             ______________________________________                                    

F. BINDING TO LECTINS

The method follows that of Example 4(G), using HB 9319 in place of HB9318. When radiolabeled lysates were preabsorbed with lentillectin-agarose beads, the antigens reactive with HB 9319 were removed.When they were preabsorbed with wheat germ agglutinin-agarose beads, theantigens were not removed. Removal of antigen was shown byimmunoprecipitation of bead supernatant, followed by SDS-PAGE.Therefore, the antigen reactive with HB 9319 characteristically binds tolentil lectin, but not to wheat germ agglutinin.

G. BIOSYNTHESIS OF HB 9319 ANTIGEN

The method is as described in Example 4(I). Pulse-chase biosyntheticstudies revealed the presence of a precursor molecule of 120 kD after a10 minute pulse with [³⁵ S]-methionine. At the 15 minute time point ofchase small amounts of the 140 kda HB 9319 antigen were also visible.Both of these molecules were observed to be present until 60 minutesafter chase. However, only the 140 kd molecule was detectable at 4 hoursafter chase until 20 hours after chase. Thus, the 120 kd componentserves as a precursor for the 140 kd component of the HB 9319 antigen.

V. EXAMPLES OF USES FOR THE PRESENT INVENTION EXAMPLE 6 THERAPEUTICTREATMENT OF HPC

Patients determined to have HPC are treated with monoclonal antibodiesreactive with HPC cells and conjugated with a toxin such as ricin, orany cytotoxic drug. The monoclonal antibody conjugates are administered(intravenously, intramuscularly, intraperitoneally, or the like, in aphysiologically acceptable carrier solution, such as phosphate bufferedsaline. The dosage is determined by the body weight of the host, itpreferably being in the range of about 0.1 mg/kg to about 40 mg/kg bodyweight, and usually about 1 mg/kg to about 10 mg/kg of host body weight.Alternatively, the dosage is established by evaluating the extent of thetumor as by quantitatively standardized ELISA, radioimaging or othermethods. Treatment is repeated at intervals as necessary, to effectenhancement of the host's ability to recover from the infection.

EXAMPLE 7 IMAGING OF HPC TUMOR

Monoclonal antibodies reactive with HPC cells are utilized to determinethe location and extent of HPC by methods well-known in the art, forexample, Larson et al. (1983) J. Clinical Investigation 72:2101, whichis incorporated by reference. Monoclonal antibodies are preferablyradiolabeled by radioiodination or by other radiolabeling techniqueswell known in the art, such as chelation using a chelating agent such asdiethylene-triaminepenta-acetic acid (DTPA); or are otherwise labeled,such as with agents having paramagnetic properties, withchemiluminescent substrates, or with components of an enzymaticreaction. The radiolabeled monoclonal antibodies are purified andformulated for pharmaceutical use. A solution of the labeled monoclonalantibodies in a carrier, for example in phosphate buffered saline, isinjected intravenously into a host. The appropriate dose is in the rangeof about 100 μg to 50 mg. Time is permitted for the antibodies tomigrate to regions of the body having concentrations of cells withantigenic determinants reactive therewith. Concentrations ofradioisotypes in certain tissues are determined or may be mapped eitherby techniques of whole body imaging which are well-known in the art,(See, for example, Rainsbury et al. (1983 ) Lancet October 22, 934(1983)) which is incorporated by reference, or by evaluating biopsiedtissue or extracted body fluid using a scintillation counter. Wherenon-radioactive labels are used, other appropriate monitoring means areemployed, such as a detector of nuclear magnetic resonance or aspectrophotometer. Areas of high radiation levels are indicative of thepresence of cells such as HPC, having the cell surface markers of thepresent invention.

The foregoing examples provide specific embodiments of the presentinvention, other embodiments being readily within the skill in the art.Thus, the scope of the present invention is defined by the followingclaims without limitation to the foregoing examples.

We claim:
 1. An isolated human heterodimeric protein complex of 400 kd by SDS PAGE or 500 kd by gel filtration which immunoreacts with Mab HB 9318 and that consists essentially of:a) a first glycopeptide characterized by having apparent molecular weight in SDS-PAGE under reducing conditions of about 205 kd and under non-reducing conditions of about 190 kd, and glycosidase susceptibility to treatment with endo F and neuraminidase but not endo H, said first glycopeptide including the amino acid residue sequence N-R-C-K-K-A-P-V-K-S-C-T-E-C-V; b) a second glycopeptide characterized by having apparent molecular weight in SDS-PAGE under reducing conditions of about 125 kd and under non-reducing conditions of about 150 kd, and glycosidase susceptibility to treatment with endo F, endo H and neuraminidase, said second glycopeptide including the amino acid residue sequence F-N-L-D-T-R-E-D-N-V-I-R-K-Y-G-D-P-G-S-L-F; and the deglycosylated and partially deglycosylated forms of said complex. 